Automated fabrication of photopatterned gelatin hydrogels for organ-on-chips applications

Abstract

Organ-on-chip platforms aim to improve preclinical models for organ-level responses to novel drug compounds. Heart-on-a-chip assays in particular require tissue engineering techniques that rely on labor-intensive photolithographic fabrication or resolution-limited 3D printing of micropatterned substrates, which limits turnover and flexibility of prototyping. We present a rapid and automated method for large scale on-demand micropatterning of gelatin hydrogels for organ-on-chip applications using a novel biocompatible laser-etching approach. Fast and automated micropatterning is achieved via photosensitization of gelatin using riboflavin-5'phosphate followed by UV laser-mediated photoablation of the gel surface in user-defined patterns only limited by the resolution of the 15 μm wide laser focal point. Using this photopatterning approach, we generated microscale surface groove and pillar structures with feature dimensions on the order of 10-30 μm. The standard deviation of feature height was 0.3 μm, demonstrating robustness and reproducibility. Importantly, the UV-patterning process is non-destructive and does not alter gelatin micromechanical properties. Furthermore, as a quality control step, UV-patterned heart chip substrates were seeded with rat or human cardiac myocytes, and we verified that the resulting cardiac tissues achieved structural organization, contractile function, and long-term viability comparable to manually patterned gelatin substrates. Start-to-finish, UV-patterning shortened the time required to design and manufacture micropatterned gelatin substrates for heart-on-chip applications by up to 60% compared to traditional lithography-based approaches, providing an important technological advance enroute to automated and continuous manufacturing of organ-on-chips.

Fig. 1. Workflow and timeline of UV-patterning approach compared to traditional micromolding

This schematic overview shows the steps required for designing and fabricating patterned gelatin substrates start-to-finish using traditional micromolding (top) or UV-patterning (bottom) fabrication. Micromolding involves a 5-time step sequence of interdependent steps, compared to a 2-time step process using UV-patterning. This 60% faster work flow is possible because UV-patterning eliminates the photolithography steps enabling separation and parallelization of work steps, such as pattern design and gelatin film fabrication.

Fig. 2. Fabrication of UV laser micropatterned hydrogels for tissue engineering

(A) Gelatin solution (gray) containing the cross-linking agent microbial transglutaminase is cast onto a COC carrier slide (blue) and allowed to cure for 12 hours. Inset: Stereomicroscope image of cured gelatin hydrogel surface. (B) Gelatin hydrogels are treated with aqueous riboflavin-5’phosphate solution (yellow), dried, and subsequently micropatterned with a 15-µm-wide UV laser beam (355 nm wavelength). (C) Hydrated and rinsed gelatin (gray) with UV laser micropatterned lines. Inset: Stereomicroscope image of micropatterned gelatin surface. Scale bars are 50 µm. (D) Addition of 0.05% riboflavin-5’phosphate to the gelatin surface. (E) UV laser etching of gelatin surface. Scale bar is 1 cm. (F) Untreated gelatin hydrogels cannot be effectively micropatterned with the UV laser engraver and instead exhibit burn marks and bubbles. Scale bar is 50 µm.

Fig. 3. Micromechanics of molded and UV laser micropatterned hydrogels

(A-C) Brightfield images of (A) micromolded (MM) gelatin lines, (B) UV micropatterned (UV-M) lines, and (C) UV micropatterned square pillars (UV-µP). Scale is 50 µm. (D-F) Contact-mode AFM topography images in 3D for (D) MM gelatin, (E) UV-M gelatin, and (F) UV-µP gelatin in liquid over an area of 40 µm² with a Z-sensor height range of 5 µm. (G) Differences in maximum and minimum Z-sensor heights (ΔZ-sensor height) for MM, UV-M, and UV-µP gelatin (n = 6–13, 2–4 samples each). *P<0.05 compared to MM gelatin by Kruskal-Wallis One Way ANOVA (H) Box and whisker plot of elastic moduli of UN, MM, UV-M, and UV-µP where a minimum of n = 75 FDCs were used for each Z-level of the pattern. The red line represents the mean, black center line represents the median, and error bars represent the 5^th and 95^th percentile. *P<0.05 compared to UN gelatin by Kruskal-Wallis One Way ANOVA

Fig. 4. Cardiac tissue engineering of neonatal rat ventricular myocytes and human iPSCs with UV laser micropatterning

(A-C) NRVMs seeded on (A) unpatterned (UN) gelatin, (B) MM gelatin, and (C) UV-M gelatin lines after 5 days in culture. Top: Bright field image. Scale is 50 µm. Bottom: Immunohistochemistry. Blue = chromatin, gray = α-actinin. Scale is 25 µm. (D-F) Human iPSCs seeded on (D) unpatterned (UN) gelatin, (E) MM gelatin, and (F) UV-M gelatin lines. Top: Bright field image. Scale is 40 µm. Bottom: Immunohistochemistry. Blue = chromatin, gray = α-actinin. Scale is 15 µm. (G) Example of intermediate OOP analysis step. Sarcomeres are detected through filtering and thresholding, and their relative orientation angles are computed using the image structure tensor. The orientation angle of each sarcomere is indicated through a color-code. Scale is 5 µm. (H) Box plot of orientational order parameter (OOP) of sarcomeric α-actinin in NRVM tissues engineered on UN (n = 3 slides, 8 images), MM (n= 4 slides, 24 images), and UV-M gels (n= 4 slides, 44 images), and in human iPSC tissues engineered on UN (n = 1 slide, 2 images), MM (n= 1 slide, 14 images), and UV-M gels (n= 2 slides, 20 images). The red line represents the mean, black center line represents the median, and bars represent 5^th and 95^th percentiles. *P < 0.05 vs. UN gelatin by Kruskal-Wallis one way ANOVA and Dunn’s Test. (I) Immunostained single compact iPSC on a UV µ-pillar island. Scale bar is 10 µm. (J) Immunostained single iPSC spread beyond the UV µ-pillar island. Green: α-actinin, blue: chromatin. Scale bar is 10 µm.

Fig. 5. Heart-on-a-chip applications of UV laser micropatterning

(A) Schematic of MTFs using UV laser micropatterning. (B) UV-M engineered MTFs: (i) Stereoscope brightfield images of engineered NRVM cardiac muscular thin films in diastole and systole (ii) after 5 days in culture. Red line indicates height of MTF detected by tracking software. Blue boxes represent initial length. Scale bar is 0.5 mm. (iii) Raw contractile stress traces at 0, 1, and 2 Hz pacing frequencies for the same representative MTF. (iv) Contractile stress of UV laser micropatterned muscular thin films (n = 9–13 films, 5–6 heart chips). Bars represent the mean ± SEM for diastolic (black), systolic (white), and twitch stress (gray). (C) Beat rate of engineered MM (black) and UV-M (red) NRVM cardiac tissues in culture over a 27 day period in beats per second. (D) Contractile stress of UV-M muscular thin films after 27 days in culture (n = 2–3 films, 1 heart chip). Bars represent the mean ± SEM for diastolic (black), systolic (white), and twitch stress (gray). *P<0.05 compared to 1 Hz pacing by Student’s T-Test.